Nghiên cứu chuyển gen vào dòng ca cao thương mại TD28 ở Việt Nam thông qua vi khuẩn Agrobacterium tumefaciens

Tgp chi Cong nghe Sinh hgc 12(2): 255-260, 2014

NGHIEN cut ) CHUYE N GEN VA O DONG C A CAO THU'ONG M^.! TD8 6

VIET NAM THON G QUA VI KHUA N AGROBACTERIUM TUMEFACIENS

Ha Hdng H^nh, Ndng Van Hii, Lg Thj Thu Hi k

Vien Nghien cmt he gen, Vien Hdn idm Khod hoc vd Cong nghe Viet Nam

Ngiy nhan bai: 02.5.2014

Ngiy nhin ding: 18.6.2014

TOM TA T

PhSt triSn ciy ca cao chuyen gen tao giong ca cao chat luong da va dang dugc quan tam tnen khai.

Trong nghien cim nay, chiing toi da thir nghiem chuyen gen vao ddng ca cao thuang mai TD8 thong qua vi

khuan Agrobucterium lumefaciens. Vector pCB30l/GUSPlus hoic pPIPRAS58 chira gen ma hda enzyme

neomycin phosphatransferase II (NPTll) va gen ma hoa enzyme p-glucuronidase (GUS/ GUSPIus) dugc sir

dyng de lay nhiem voi cac manh mo ca cao co ngudn gdc tu phoi soma so cap. Sau 2 ngay ddng nuoi cay, cac

manh phoi soma dugc tai sinh tren moi Iruong phat trien md seo thii cap (SCO) chiia 20 g/l glucose, 2 mg/l

2,4D, 0,05 mg/l BAP, cd bo sung 50 mg/l kanamycln cho chpn loc NPTll va 200 mg/l moxalactam de loai bo

A. lumefaciens. Mo seo dugc chuyen sang qua trinh t^io phdi tren mdi trudng cam irng t^o phoi ED chiia 30 g/l

sucrose, 1 g/l glucose, 50 mg/I kanamycin, 200 mg/I moxalactam. Cac phoi soma thii cap chuyen gen dugc tao

choi tren moi tradng EDL c6 chiia 0,3 g/l KNO3, 1 ml/1 amino acid, 10 g/I sucrose va 20 g/l glucose, 50 mg/i

kanamycin, 200 mg/l moxalactam va hinh thanh re tren mdi tmong RD cd 0,3 g/l KNOj, 5 g/l sucrose, !0 g/l

glucose, 50 mg/I kanamycin, 200 mg/l moxalactam. Ket qua phan tich cay chuyen gen thong qua bieu hien cua

gen GUS va kiem ga bang PCR vdi gen NPTll da khang dmh sy co mat ciia gen dugc chuyen trong cay ca cao

chuyen gen.

Tit khda' Agrobacterium lumefaciens. co coo, chuyen gen. ddng TDS, gen GUS/ GUSPIus, phoi soma

MO DAU

Ciy ca cao (Theobroma cacao L.) la cay cdng

nghiep cd gia tri kinh te cao, cd ngudn gdc tir Trang

Nam My, hien dang dugc trdng phd bien d khap cie

nudc cd khi hau nhiet ddi ndng im. O Viet Nam, ca

cao dugc du nhap vio rit sdm, den nam 2013, dien

tich trdng ca cao la 22 110 ha, trong dd viing cd dien

tich trdng ldn nhit d ddng bang sdng Cuu Long vi

Tay Nguyen. Dien tich ca cao thu hoach khoing

11.055 ha, chi^m 50% tdng dien tich, sin lugng hat

ca cao khd len men nam 2013 la 6.765 tin, tang nhe

so vdi nim 2012 (65 tin), trong dd phin ldn ca cao

dugc xuit khiu (http://www.kliuvennonp.vn/). Tuy

nhien, sin lugng ca cao hing nam bi inh hudng nang

ne hdl sau b?nh v i nam hai ciy.

Cimg vdi nhieu ciy trdng ndng nghiep v i mgt so

cay cdng nghiep chinh, ca cao dang li ddi tugng

dugc quan tim trong cai bien di truyen phuc vu

nghien ciiu he gen hgc chitc ning, tien tdi iing dyng

chgn tao gidng ting hem niia nang suit, chit luong

vi tao kha ning chdng chm b?nh, thich nghi vdi cie

dieu kien sinh thai. Tuy nhien, kha nang tai sinh in

vitro v i hieu qui chuyen gen vio ca cao khdng cao,

phu thudc nhieu vio kieu gen. De thiet lap vi tdi uu

hda quy trinh chuyen gen vio ca cao, mgt sd nghien

ciiu d i sii dung cie gen chi thi nhu gen mi hda

protein phat huynh quang (Enhanced Green

Fluorescent Protein - EGFP) va gen chi thi nhudm

miu md te bio p-glucuronidase (GUS) ket hgp he

thdng tai smh in vitro tir phdi soma thdng qua vi

khuin A. lumefaciens. Nam 2003, Maximova vi

ddng tic gii d i md ti quy trinh chuyen gen EGFP

vao ca cao thdng qua vi khuin A. lumefaciens chimg

AGL I. Gan day, Silva vi ddng tie gii (2009) da

nhin thiy inh hudng tich cue cua cie polyamine vi

khing smh diet khuan P-lactam den qui trinh phit

smh phdi soma trong qui trinh chuyen gen GUS vao

ddng ca cao TSH 565. Tuy nhien, nghien ciru niy

mdi chi dirng d giai doan kiem tra cac phdi soma sau

chuyen gen, chua den giai doan tao cay hoan chinh.

Trong cac nghien cim trudc diy, chiing tdi da

thinh cdng trong xay dyng quy trinh tai sinh tn vitro

mgt sd ddng ca cao thuong mai d Viet Nam thdng

qua phdi soma (Hi Hdng Wa.ah etal, 2013a, 2013b).

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